Cross-Resistance Among Next-Generation Antiandrogen Drugs Through the AKR1C3/AR-V7 Axis in Advanced Prostate Cancer

Abstract

The next-generation antiandrogen drugs, XTANDI (enzalutamide), ZYTIGA (abiraterone acetate), ERLEADA (apalutamide) and NUBEQA (darolutamide) extend survival times and improve quality of life in patients with advanced prostate cancer. Despite these advances, resistance occurs frequently and there is currently no definitive cure for castration-resistant prostate cancer. Our previous studies identified that similar mechanisms of resistance to enzalutamide or abiraterone occur following treatment and cross-resistance exists between these therapies in advanced prostate cancer. Here, we show that enzalutamide- and abiraterone-resistant prostate cancer cells are further cross-resistant to apalutamide and darolutamide. Mechanistically, we have determined that the AKR1C3/AR-V7 axis confers this cross-resistance. Knockdown of AR-V7 in enzalutamide-resistant cells resensitize cells to apalutamide and darolutamide treatment. Furthermore, targeting AKR1C3 resensitizes resistant cells to apalutamide and darolutamide treatment through AR-V7 inhibition. Chronic apalutamide treatment in C4-2B cells activates the steroid hormone biosynthesis pathway and increases AKR1C3 expression, which confers resistance to enzalutamide, abiraterone, and darolutamide. In conclusion, our results suggest that apalutamide and darolutamide share similar resistant mechanisms with enzalutamide and abiraterone. The AKR1C3/AR-V7 complex confers cross-resistance to second-generation androgen receptor-targeted therapies in advanced prostate cancer.

Figure 1.. Enzalutamide/abiraterone resistant prostate cancer cells are cross-resistant to apalutamide and darolutamide.

A. C4-2B parental, C4-2B AbiR, C4-2B MDVR and CWR22Rv1 cells were treated with different concentrations of enzalutamide, apalutamide or darolutamide for 3 days. Total cell numbers were counted and cell survival rate was calculated. B. C4-2B parental, C4-2B MDVR, C4-2B AbiR and CWR22Rv1 cells were treated with 20 μM enzalutamide, 20μM apalutamide or 5 μM darolutamide for 0, 3 and 5 days and total cell numbers were determined. C. The clonogenic ability of C4-2B parental, C4-2B MDVR, C4-2B AbiR and CWR22Rv1 cells treated with 20 μM enzalutamide, 20 μM apalutamide or 5 μM darolutamide was analyzed. Enza: enzalutamide, APAL: apalutamide, Abi: abiraterone acetate, DARO: darolutamide. * p<0.05.

Figure 2.. AR-V7 confers apalutamide and darolutamide resistance.

A. C4-2B parental, C4-2B MDVR and CWR22Rv1 cells were treated with 20 μM enzalutamide, 20 μM apalutamide or 5 μM darolutamide for 3 days. Whole cell lysates were collected and subjected to western blot. B. C4-2B cells were transfected with PSA luciferase promoter in CS-FBS condition for 24 hours, followed by treatment with 1 nM DHT with or without 20µM enzalutamide, 20 μM apalutamide, 5, 10 or 20 μM darolutamide or 0.5 μM niclosamide overnight. Whole cell lysates were collected and subjected to luciferase assay (top). C4-2B cells were co-transfected with PSA luciferase promoter and AR-V7 in CS-FBS conditions for 24 hours, followed by treatment with 20µM enzalutamide, 20 μM apalutamide, 5, 10 or 20 μM darolutamide or 0.5 μM niclosamide overnight. Whole cell lysates were collected and subjected to luciferase assay (bottom). C. C4-2B MDVR cells were transiently transfected with control siRNA or AR-V7 siRNA. Following transfection, cells were treated with 20 µM apalutamide or 5 μM darolutamide and cell numbers were determined after 5 days. D. Whole cell lysates were collected and subjected to western blot. E. CWR22Rv1 cells were transiently transfected with control siRNA or AR-V7 siRNA. Following transfection, cells were treated with 20 µM apalutamide or 5 μM darolutamide and cell numbers were determined after 5 days. F. Whole cell lysates were collected and subjected to western blot. Enza: enzalutamide, APAL: apalutamide, Abi: abiraterone acetate, DARO: darolutamide. NIC: niclosamide. n.s.: not-significant. * p<0.05

Figure 3.. AKR1C3 confers apalutamide and darolutamide resistance through AR-V7 regulation.

A-B. C4-2B MDVR cells were infected with lenti-control shRNA or lenti-AKR1C3 shRNA and then treated with 20 µM apalutamide or 5 μM darolutamide and cell numbers were determined after 5 days. Whole cell lysates were collected and subjected to western blot. C-D. CWR22Rv1 cells were infected with lenti-control shRNA or lenti-AKR1C3 shRNA and then treated with 20 µM apalutamide or 5 μM darolutamide and cell numbers were determined after 5 days. Whole cell lysates were collected and subjected to western blot. E-F. C4-2B neo or C4-2B AKR1C3 cells were treated with different concentrations of apalutamide or darolutamide for 3 days, total cell numbers were counted and cell survival rate (%) was calculated. Whole cell lysates were collected and subjected to western blot. G. C4-2B MDVR and CWR22Rv1 cells were treated with 20 µM apalutamide or 5 μM darolutamide or 20 µM indomethacin, alone or in combination, total cell numbers were determined on 3 and 5 days. APAL: apalutamide, DARO: darolutamide. INDO: indomethacin * p<0.05.

Figure 4.. Chronic apalutamide treatment in C4-2B cells activates the steroid biosynthesis pathway through AKR1C3 upregulation.

A. C4-2B parental and C4-2B APALR cells were treated with different concentration of apalutamide for 3 days, total cell numbers were determined and cell survival rate was calculated. B. C4-2B parental and C4-2B APALR cells were treated with 20 μM apalutamide, 5 μM darolutamide, 20 μM enzalutamide or 10 μM abiraterone and cell numbers were determined after 3 days. C-D. The clonogenic ability of C4-2B parental and C4-2B APALR cells treated with 20 μM apalutamide, 5 μM darolutamide, 20 μM enzalutamide or 10 μM abiraterone was analyzed. E. The genes involved in steroid hormone biosynthesis pathway were determined by qRT-PCR. F. Whole cell lysates were collected from C4-2B parental, C4-2B MDVR and C4-2B ARNR cells and subjected to western blot. Enza: enzalutamide, APAL: apalutamide, Abi: abiraterone acetate, DARO: darolutamide. * p<0.05

Figure 5.. Targeting AKR1C3 resensitizes apalutamide resistant cells to apalutamide and enzalutamide.

A. C4-2B APALR cells were treated with 20 µM enzalutamide, 20 µM apalutamide or infected with lenti-AKR1C3 shRNA, alone or in combination. Total cell numbers were determined on 3 and 5 days. B. C4-2B APALR cells were infected with lenti-control shRNA or lenti-AKR1C3 shRNA and then treated with 20 µM enzalutamide or 20 µM apalutamide. After 5 days, whole cell lysates were collected and subjected to western blot. C. C4-2B APALR cells treated with 0, 20, 40 µM indomethacin with or without 20 μM apalutamide and cell numbers were determined after 5 days. D-E. The clonogenic ability of C4-2B ARNR cells treated with 0, 20, 40 µM indomethacin with or without 20 μM apalutamide was analyzed. APAL: apalutamide, ENZA: enzalutamide. INDO: indomethacin * p<0.05.